yS-T cells are a rare subpopulation of T lymphocytes with unique antitumor properties. Cancerous cells -particularly those of epithelial origin - are nowknown to display a variety of stress-induced antigens (such as MICA/B), which can serve as recognition determinants for the human y8-T cell receptor (TCR) and associated accessory molecules. Thus, in order to kill tumor cells, y8-T cells do not require the presence of tumor-specific antigens, tumor antigen processing or major histocompatibility complex (MHC) display of tumor-specific peptides. Given this, we propose that y8-T cells are ideal to study in the context of developing novel approaches for the primary or adjuvant treatment of a number of common human cancers of epithelial origin - including breast cancer. Nevertheless, despite the theoretical promise of y8-T cell-based immunotherapy, to date clinical studies to examine this question are only now in the earliest stages of development. Indeed, until very recently, no practical means existed to generate the large numbers of y8-T cells required for clinical-scale administration. Rationale: We initially identified a CD2-initiated signaling pathway which inhibits apoptosis in mitogen-stimulated human y8-T cells. By exploiting this signaling pathway, our laboratory has pioneered the development of the methods permitting the large-scale ex vivo expansion of human y8-T cells. Importantly, these y8-T cells retain potent innate antitumor activity in vitro against a variety of human tumor cell lines, including breast cancer cell lines. In addition, our preliminary studies demonstrate that when administered intravenously, these y5-T cells can inhibit the growth of human breast cancer tumors established in nude mice. Together, these findings provide both the biological and clinical rationale for the studies proposed in this grant. Approach: The aims of this proposal are as follows. Aim 1: To characterize the y8-T cell population present in breast cancer patients in regard to numbers in the circulation, their ability to be expanded ex vivo and their cytotoxicity to breast cancer cells. Aim 2: To optimize and standardize the large-scale expansion and purification of human y5-T cells for subsequent use in clinical trials. Studies from this aim will yield an IND application to be filed with the US FDA. Aim 3: To examine and optimize expanded human yS-T cell immunotherapy in a human xenogeneic breast cancer model. Aim 4: To initiate clinical trials of y8-T cell immunotherapy in patients with therapy-resistant metastatic breast cancer. Long-term objectives: We propose to follow our first-generation trial with subsequent Phase I studies that will combine the y5-T cell regimen with immunomodulatoryagents and/or chemotherapy based on observations made in our animal models. These studies would then form the basis for future Phase II trials incorporating such treatment strategies. Lay description: y8-T cells are a rare type of blood cell which can naturally kill cancer cells making them ideal to study as a new form of cancer therapy. However, they are extremely difficult to grow in the laboratory. As we have discovered a means to generate large numbers of these cells, we are now able to propose clinical studies designed to examine how these rare tumor-killing cells might be used as a newform of treatment for breast cancer - or other types of cancers.